CN106868224A - The RT PCR methods of detection pig blue-ear disease poison classical strainses, highly pathogenic mutant strain and the strains of NADC 30 - Google Patents
The RT PCR methods of detection pig blue-ear disease poison classical strainses, highly pathogenic mutant strain and the strains of NADC 30 Download PDFInfo
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Abstract
The invention discloses a kind of RT PCR methods for detecting pig blue-ear disease poison classical strainses, highly pathogenic mutant strain and the strains of NADC 30, the present invention with reference to porcine reproductive and respiratory syndrome virus (PRRSV) the Nsp2 gene orders delivered in GenBank, the pair of primers using Primer5.0 Software for Design.This can be used for the different hypotype of RT PCR methods detection pig blue-ear disease three kinds of amino acid numbers of poison to primer.Sensitivity of the invention is high, specificity is good, pig blue-ear disease poison three kinds of hypotype-classical strainses, highly pathogenic mutant strain and the strains of NADC 30 can successfully be detected, the doubtful clinical sample with pig blue-ear disease poison can be detected, so that for the diagnosis and epidemiological surveillance of pig blue-ear disease poison.
Description
Technical field
The invention belongs to technical field of molecular biology, specifically, be related to a kind of detection pig blue-ear disease poison classical strainses,
The RT-PCR method of highly pathogenic mutant strain and NADC-30 strains.
Background technology
Porcine reproductive and respiratory syndrome virus (PRRSV) are also called pig blue-ear disease, to cause Sow abortion, produce stillborn foetus, weak
Tire, mummy tire and piglet and growing and fattening pigs expiratory dyspnea, septicemia, high mortality are principal character.The disease is all over the world
Generally existing, serious economic loss is caused to the pig industry of China.In recent years, the variation phenomenon of PRRSV causes people's
Concern, external research shows there is obvious sequence difference between the isolated strain of same gene type, and the variation of PRRSV is
Cause one of this sick unmanageable major reason.
At present in the world according to the antigenic characteristic of PRRSV, 2 serotypes of Europe class and american type, America can be classified as
Type strain is divided into three kinds of hypotypes according to it in the missing of Non-structural protein NSP2 base number again, and respectively classical strainses, height causes a disease
Property variation strain and NADC-30 strains.
RT-PCR method concrete operation step is specific fragment gene first with primer amplified sample nucleic, is taken
PCR primer runs agarose gel electrophoresis, is then taken pictures observation in gel imaging instrument, and foundation genes of interest clip size judges whether
It is NADC-30 target fragments, such as genetic fragment size is 493bp, is judged to that pig blue-ear disease poison is positive, such as genetic fragment size is
860bp, then be judged to that highly pathogenic PRRS poison is positive, if genetic fragment size is 757bp, is judged to pig blue-ear disease
Malicious class NADC-30 strains are positive, are otherwise judged to feminine gender.RT-PCR method specificity and sensitivity are high.
Porcine reproductive and respiratory syndrome virus (PRRSV) three kinds of hypotypes are detected simultaneously without a kind of in currently available technology
The RT-PCR method of (classical strainses, highly pathogenic mutant strain, NADC-30 strains).Expanded with PCR respectively using single pair of primers
Increase the shortcomings of three kinds of hypotypes have complex operation, detection time is long, it is impossible to understand the genetic traits of detection sample PRRS in time.
The content of the invention
In view of this, with PCR three kinds of hypotypes of amplification there is complex operation, inspection in the present invention respectively for using single pair of primers
Survey time problem long, there is provided one kind detection pig blue-ear disease poison classical strainses, highly pathogenic mutant strain, NADC-30 strains
RT-PCR method, can to it is doubtful with pig blue-ear disease poison clinical sample detect so that for pig blue-ear disease poison
Diagnosis and epidemiological surveillance.The reproductive and respiratory syndrome virus of mesh first three different subtype generally existing in swinery, but do not have inspection simultaneously
Survey three kinds of detection methods of the reproductive and respiratory syndrome virus of hypotype.The present invention on the basis of many plants of PRRSV Nsp2 gene orders are compared, if
A pair are counted and can distinguish classical strainses, the specificity of three kinds of different genotype PRRSV of highly pathogenic strain and NADC30 strains and drawn
Thing, the present invention is a kind of simple, quick, sensitivity strong detection method high and special.
In order to solve the above-mentioned technical problem, the invention discloses one kind detection pig blue-ear disease poison classical strainses, variant and
The primer of NADC-30 sample strains, for RT-PCR reactions, the primer includes sense primer and anti-sense primer, the sense primer
With the nucleotide sequence of anti-sense primer respectively such as SEQ ID NO:1 and SEQ ID NO:Shown in 2.
The present invention also provides a kind of detection pig blue-ear disease poison classical strainses, highly pathogenic mutant strain and NADC-30 strains
RT-PCR method, comprise the following steps：As template, reverse transcription obtains cDNA to RNA with detected sample, is drawn with above-mentioned
Thing, RT-PCR amplifications are carried out to sample to be tested, and amplified production is detected with 1.5% agarose gel electrophoresis；Amplified production size is
887bp, then contain in sample or candidate contains pig blue-ear disease poison classical strainses；Amplified production size is 797bp, then contain in sample
Have or candidate contains highly pathogenic mutant strain；Amplified production size is 493bp, then contain in sample or candidate contains NADC-
30 strains.
Further, reverse transcription is carried out with the reverse transcription reagent box purchased from precious bioengineering (Dalian) Co., Ltd, it is described
The step of reverse transcription is：By the μ L of RNA templates 4 and No. 1 the μ L of 5 × Prime Script Buffer 4, No. 2 Prime ScriptRT
The μ L of Enzyme Mix 1, No. 4 μ L of Random 6mers 2 and No. 5 RNase Free dH2The μ L of O 9 are mixed, and are put into PCR instrument enterprising
Row reverse transcription.
Further, the reaction condition of reverse transcription is：37 DEG C of 15min, 85 DEG C of 15s, 16 DEG C of 10min.
Further, the reaction system of the RT-PCR amplifications is as follows：μ L, the Quick Taq HS of template cDNA 3
The μ L of DyeMix 10, sense primer and anti-sense primer each 0.6 μ L, it is remaining by ddH2O is supplied, and total amount is 20 μ L.
Further, the reaction system condition of RT-PCR amplifications is as follows：94 DEG C of predegeneration 2min；94 DEG C are denatured 30s, 55 DEG C
Annealing 30s, 68 DEG C of extension 1min, totally 35 circulations；72 DEG C of extension 8min.
Compared with prior art, the present invention can be obtained including following technique effect：
1) the method sample of nucleic acid with PRRSV doubtful to 200 parts that the present invention sets up carries out RT-PCR detections, recall rate
It is 46.5% (93/200), wherein classical strainses verification and measurement ratio is that 8.5% (17/200), highly pathogenic mutant strain recall rate is
28.5% (57/200) and NADC-30 strains recall rate be 9.5% (19/200).
2) present invention has reliability higher, and specific good, sensitivity is high, overcomes and three kinds of strains are examined respectively
What survey was brought takes, consumes reagent, the risk of increase contamination probability, and diagnosis and preventing and treating to PRRSV compared with kit have
More directly and effective meaning.
3) not only operating procedure is few for the present invention, can save the plenty of time, than detection PRRSV substances or duplex RT-PCR method
It is sensitiveer, quick, it is that the quick diagnosis of pig blue-ear disease and epidemiology survey are laid a good foundation.
Certainly, implement any product of the invention to it is not absolutely required to while reaching all the above technique effect.
Brief description of the drawings
Accompanying drawing described herein is used for providing a further understanding of the present invention, constitutes a part of the invention, this hair
Bright schematic description and description does not constitute inappropriate limitation of the present invention for explaining the present invention.In the accompanying drawings：
Fig. 1 is 1.5% agarose after positives template of the invention is expanded through 55 DEG C of -61 DEG C of 11 annealing temperature RT-PCR
Gel electrophoresis figure, wherein, M representation DNAs standard II, N represents negative control, and 1-7 represents experiment sample, respectively 55 DEG C, 56 DEG C,
57 DEG C, 58 DEG C, 59 DEG C, 60 DEG C and 61 DEG C, wherein 57 DEG C is most suitable annealing temperature；
Fig. 2 is RT-PCR amplifications after positives template of the invention mixes with the μ L-1 μ L of various concentrations upstream and downstream primer 0.2
Afterwards, 1.5% agarose gel electrophoresis figure；Wherein, M representation DNAs standard II, N represents negative control, 1-5 swimming lanes be with 0.2 μ L,
0.4 μ L, 0.6 μ L, 0.8 μ L, the 1 μ mixed experiment samples of L concentration, wherein 0.6 μ L are optimal primer concentration；
Fig. 3 is positives template sterilized water of the invention from 10-1To 10-9After doing after 10 times of serial dilutions through RT-PCR,
1.5% agarose gel electrophoresis figure；Wherein, M representation DNAs standard II, N represents negative control, and positive template is dilute successively for 1-5 swimming lanes
Releasing multiple is：1.0×10-3、1.0×10-4、1.0×10-5、1.0×10-6With 1.0 × 10-7。
Specific embodiment
Describe embodiments of the present invention in detail below in conjunction with embodiment, thereby to the present invention how application technology hand
Section can fully understand and implement according to this to solve technical problem and reach the implementation process of technology effect.
The design of primers of embodiment 1
Three subtype sequences (pig blue-ear disease poison classical strainses (EF536003), the height of the PRRSV genes logged according to NCBI
Pathogenicity variation strain (EF112445) and NADC-30 strains (JN654459)), it is directed to PRRSV using Primer5.0 Software for Design
The use of Nsp2 genes can distinguish pig blue-ear disease poison classical strainses, highly pathogenic mutant strain and NADC-30 strains specificity
Primer, all primers synthesize by Shanghai Sheng Gong bioengineering Co., Ltd.
Sense primer (Nsp2F primers)：GACACCTCCTTTGATTGG, SEQ ID NO:1；
Anti-sense primer (Nsp2R primers)：GAGAGGAIGCAGACAAATC (wherein I represents T or C or A or G), SEQ ID
NO:2；
Expanded using above-mentioned primer RT-PCR, can be by the size of agarose gel electrophoresis band to three kinds of PRRSV
Hypotype is efficiently differentiated differentiation, and specificity and sensitivity are high, Cao Zuo Jian Unit.
The RT-PCR side of the detection pig blue-ear disease poison of embodiment 2 classical strainses, highly pathogenic mutant strain and NADC-30 strains
Method
The method is comprised the following steps：
(1) measuring samples RNA is extracted：
Blood sample to be checked is centrifuged 5 minutes by 8000g, takes upper serum and respectively uses；Histoorgan sample is needed with 1:5
Mass volume ratio mixes with sterilizing distilled water, and 8000g is centrifuged 5 minutes after grinding, takes supernatant standby.
(2) RNA for extracting is added to and is inverted purchased from the reverse transcription reagent box of precious bioengineering (Dalian) Co., Ltd
Record, obtains cDNA templates：
Reverse transcription step is：By the μ L of RNA templates 4 and No. 1 μ L of 5 × PrimeScript Buffer 4, No. 2
The μ L of PrimeScriptRT Enzyme Mix 1, No. 4 μ L of Random 6mers 2 and No. 5 RNase Free dH2The μ L of O 9 are mixed,
Being put into carries out reverse transcription in PCR instrument.Its reaction condition：37 DEG C of 15min, 85 DEG C of 15s, 16 DEG C of 10min.This reverse transcription reagent box is purchased
From Dalian treasured bioengineering Co., Ltd.
(3) RT-PCR steps
PCR presses 20 μ L reaction systems：The μ L of 3 μ L, Quick Taq HS DyeMix of template cDNA 10, upstream and downstream primer is each
0.6 μ L, it is remaining by ddH2O is supplied.Nsp2F/R primer reaction conditions：94 DEG C of predegeneration 2min；94 DEG C of denaturation 30s, 55 DEG C are moved back
Fiery 30s, 68 DEG C of extension 1min, totally 35 circulations；72 DEG C of extension 8min.The PCR primer for obtaining takes 5 μ L, 1.5% agaroses and coagulates
Gel electrophoresis are observed.
(4) testing result：Amplified production size is 887bp (SEQ ID NO:3), then in sample contain or candidate contain through
The pig blue-ear disease poison of allusion quotation strain；Amplified production size is 797bp (SEQ ID NO:4), then contain in sample or candidate contains height
The pig blue-ear disease poison of pathogenicity variation strain；Amplified production size is 493bp (SEQ ID NO:5), then contain or wait in sample
Select the pig blue-ear disease poison containing NADC-30 strains.
The measure of optimization and the sensitivity of the annealing temperature of embodiment 3 and primer concentration
(1) Cloning Transformation and restructuring positive plasmid：RT-PCR amplifies genes of interest, carries out electrophoresis observation and analysis.Electrophoresis
The positive sample for screening is purified with carrying out PCR primer purchased from OMGA companies PCR purification kits, the PCR primer that will be purified
It is connected with TMD-19T carriers, is taken out after being put into metal bath 12h.The connection product for obtaining adds competent cell (Escherichia coli
DH5 α plants) afterwards liquid add 1mL LB liquid in, be put into 37 DEG C and shake bacterium case, then 5000r/min 4min centrifugation, abandon on 800 μ L
Clear liquid, is left 200 μ L piping and druming and mixes.Absorption bacterium solution drips the culture medium in LB+AMP, and bacterium solution is uniformly distributed with spreading rod
On culture medium, 37 DEG C of bacteriological incubator 12h~16h are put.Picking single bacterium colony is mixed in being dissolved in 10 μ L aqua sterilisas, takes 2 μ L bacterium solutions
For pcr template runs electrophoresis.The bacterium solution of picking bright wisp band shakes bacterium 12h in being added to 4mL LB+AMP liquid.With the plasmid of OMGA companies
Extracts kit carries out the extraction of plasmid to the bacterium solution shaken, and the plasmid for obtaining i.e. positive template puts -20 DEG C of preservations.
(2) optimization of annealing temperature：Annealing temperature in Nsp2F/R primer reaction conditions sets 55 DEG C -65 DEG C, obtains
PCR primer takes 5 μ L and is observed with 1.5% agarose gel electrophoresis.The temperature for choosing most bright wisp band is optimum annealing temperature, such as
Shown in Fig. 1, optimum annealing temperature of the invention is 55 DEG C.
(3) optimization of primer concentration：PCR presses 20 μ L reaction systems, each 0.2 μ L-1 μ L of upstream and downstream primer.Choose most bright wisp
The primer concentration of band is optimal primer concentration, as shown in Fig. 2 optimal primer concentration is 0.6 μ L.
(4) the above-mentioned positive template sterilized water for preparing is done into 10 times of serial dilutions：10-1-10-7, take 1.0 × 10-3、1.0×10-4、1.0×10-5、1.0×10-6With 1.0 × 10-7The plasmid of dilution, the optimal RT-PCR reaction systems more than
Expanded with reaction condition, the PCR primer for obtaining takes 5 μ L and observed with 1.5% agarose gel electrophoresis, detects that its is minimum
Concentration is shown in as shown in figure 3, the RT-PCR reaction detection limits set up are respectively：Classical strainses -1.13 × 104Copy number/
Reaction, highly pathogenic mutant strain -9.47 × 103Copy number/reaction, NADC-30 strains -2.30 × 104Copy number/reaction, table
Bright sensitivity is good.
Comparative example 1
Differentiate that porcine reproductive and respiratory syndrome virus are highly pathogenic and pass through according to the national standard GB/T 27517-2011 for having reported
Allusion quotation strain multiplex RT -PCR method, the primer in component state standard, using the method core with PRRSV doubtful to 200 parts
Acid sample is detected：The RNA of test serum is extracted with TriZol methods, RNA ddH are obtained2- 20 DEG C of refrigerators are put after O dissolvings to protect
Deposit.RT- is carried out to the RNA for extracting with the reverse transcription reagent box purchased from Co., Ltd of precious Shanghai Qiao Yu bio tech ltd
PCR is expanded, and reaction system is 20 μ L：Wherein often pipe adds the μ L of 10 μ L 5 × Reaction of RNA, M-MLV Buffer 4,
The 0.5 μ L of μ L, M-MLV of 2.5mmol/L dNTPs 4, RNase inhibitor carries 0.5 μ L, composite PCR share reverse transcription primer (5 '-
GAGCTGAGTATTTTGGGCGTG-3 ', such as SEQ ID NO:Shown in 8) 1 μ L.37 DEG C of 1h in PCR instrument are put into, after 70 DEG C of 15min
It is directly used in following PCR amplifications；Composite PCR reaction system composition：The μ L of 4 μ L, 10 × PCR buffer solutions of cDAN 2.5,
The μ L of 2.5mmol/L dNTPs 2, sense primer (5 '-GGTTCGGAAGAAACTGTCGG-3 ', such as SEQ ID NO:Shown in 6) 0.5
μ L, sense primer (5 '-AGCAGGTGGAAGAAGCGAATC-3 ', such as SEQ ID NO:Shown in 7) 0.5 μ L, anti-sense primer (5 '-
GAGCTGAGTATTTTGGGCGTG-3 ', such as SEQ ID NO:Shown in 8) 1 μ L, the shared μ L of reverse transcription primer 1 of composite PCR.Wherein
Reagent is purchased from precious bioengineering (Dalian) Co., Ltd.Response procedures are as follows：94 DEG C of 45s, 59 DEG C of 45s, 70 DEG C of 45s, totally 35
Individual circulation；70 DEG C of extension 10min.1% agarose gel electrophoresis detects amplification, wherein recall rate 48.5%, classical strainses
Verification and measurement ratio is that 9% (18/200), highly pathogenic mutant strain recall rate is 29.5% (59/200).
The method sample of nucleic acid with PRRSV doubtful to 200 parts that the present invention sets up carries out RT-PCR detections, and recall rate is
46.5% (93/200), wherein classical strainses verification and measurement ratio is that 8.5% (17/200), highly pathogenic mutant strain recall rate is
28.5% (57/200) and NADC-30 strains recall rate be 9.5% (19/200)；The inventive method is with the coincidence rate of the method
95.9%.
Comparative example 2 is purchased from Anheal Laboratories Co., Ltd porcine reproductive and respiratory syndrome virus RT-
PCR diagnostic kits (universal)
Serum sample and yin and yang attribute are compareed into each 100 μ L to be placed in 1.5mL sterile centrifugation tubes, the μ of lysate 600 is separately added into
L is stored at room temperature 3~5min after mixing.Draw liquid into the adsorption column for being cased with collecting pipe, 13000rmp centrifugation 30s discard receipts
Liquid in collector, liquid (repetition two in collecting pipe is discarded to 600 μ L cleaning solutions, 13000rmp centrifugation 30s are added in adsorption column
It is secondary).Void column centrifugation 13000rmp centrifugation 30s, during adsorption column moved into new 1.5mL centrifuge tubes, add 25 μ L eluents, room again
Temperature stands 1min, and 13000rmp centrifugations 30s obtains total serum IgE.RT-PCR reaction systems：μ L, 16.8 μ L RT-PCR are anti-for cumulative volume 20
Answer liquid, 1.2 μ L enzyme mixations, 2 μ L template ribonucleic acids.Program is carried out on PCR amplification instrument device：42℃45min,94℃3min；
Amplification condition：94 DEG C of 30s, 55 DEG C of 30s, 72 DEG C of 30s, totally 35 circulations；72 DEG C of extension 7min.2% agarose gel electrophoresis is examined
Survey amplification.
The classical strainses recall rate of (universal) detection of porcine reproductive and respiratory syndrome virus RT-PCR diagnostic kits is
50.5% (101/200), reach 92.1% with the inventive method result coincidence rate.
The porcine reproductive and respiratory syndrome virus of comparative example 3 (variants of Nsp2 1594~1680) RT-PCR detection kit
The experimental procedure of the kit is with comparative example 2.
The height of porcine reproductive and respiratory syndrome virus (variants of Nsp2 1594~1680) RT-PCR detection kit detection
Pathogenicity variation strain recall rate is 28% (56/200), and 98.2% is reached with the inventive method result coincidence rate.
Can be drawn from comparative example 1-3：Illustrate that the present invention has reliability higher, specific good, sensitivity is high, overcomes
Three kinds of strains are detected with time-consuming, the consumption reagent for being brought, the risk for increasing contamination probability respectively, compared with kit,
Diagnosis and preventing and treating of the present invention to PRRSV have more direct and effective meaning.
The application examples of embodiment 4
The lungs of the doubtful piglet with blue otopathy, groin are gathered from Mianyang, Sichuan, Guangyuan, Beichuan and Deyang and other places to drench
Fawn on 150 parts of serum equal samples, extract RNA and reverse transcription into cDNA in sample.According to the RT-PCR method of present invention report
Amplification system and amplification condition are detected, and set to blue otopathy classical strainses, highly pathogenic mutant strain, NADC-30 strains
Vertical yin and yang attribute control, takes result of determination after the μ L of PCR primer 5 carry out electrophoresis in 1.5% agarose gel electrophoresis：PRRSV is detected
Rate is 37.3% (56/150), and wherein PRRSV classical strainses positive rate is 8% (12/150)；PRRSV highly pathogenic mutant strains
Positive rate is 16.7 (25/150) %；PRRSV NADC30 strains positive rate is 12.6% (19/150).Through with comparative example 1-3's
Three kinds of methods reported, primer (GBT27517-2011) and purchase kit in component state standard (are purchased from Beijing Century
Yuan Heng animal epidemic preventions Technology Co., Ltd.) detect that the PCR testing results of these three PRRSV strains are compared analysis coincidence rate and are
91.6~100% (such as tables 1).
The inventive method of table 1 compares clinical pathological material of disease testing result with report method
Described above has shown and described some preferred embodiments of invention, but as previously described, it should be understood that invention is not
Form disclosed herein is confined to, the exclusion to other embodiment is not to be taken as, and can be used for various other combinations, modification
And environment, and can be carried out by the technology or knowledge of above-mentioned teaching or association area in invention contemplated scope described herein
Change.And the change and change that those skilled in the art are carried out do not depart from the spirit and scope of invention, then all should be in the appended power of invention
In the protection domain that profit is required.
SEQUENCE LISTING
<110>Southwest University for Nationalities
<120>The RT-PCR method of detection pig blue-ear disease poison classical strainses, highly pathogenic mutant strain and NADC-30 strains
<130> 2017
<160> 8
<170> PatentIn version 3.3
<210> 1
<211> 18
<212> DNA
<213>Artificial sequence
<400> 1
gacacctcct ttgattgg 18
<210> 2
<211> 19
<212> DNA
<213>Artificial sequence
<400> 2
gagaggatgc agacaaatc 19
<210> 3
<211> 886
<212> DNA
<213>PRRSV genes
<400> 3
gacacctcct ttgattggaa tgttgtgctt cctggggttg aggcggcgaa tcagacaacc 60
aaacagctcc acgtcaacca gtgccgcgct ctggctcctg tcgtgactca agagcctttg 120
gacaaagact cggtccctct gaccgccttc tcgctgtcca attgctatta ccctgcacaa 180
ggtgacgagg ttcgtcaccg tgagaggtta aattccgtac tctctaagtt ggagggggtt 240
gttctagaag aatatgggct catgtccact ggacttggcc cgcgacccgt actgccgagc 300
gggctcgacg agcttaaaga ccagatggag gaggatctgc tagaactagc caacacccag 360
gcgacttcag aaatgatggc ctgggcggcc gagcaggttg atttaaaagc ttgggtcaaa 420
agctacccac ggtggacacc accaccccct ccaccaagag ttcaacctcg aaaaacgaag 480
cctgtcaaga gcttgccaga gaacaagcct gtccccgctc cgcgcaggaa ggttagatcc 540
gactgtggca gcccgatttt aatgggcgac aatgtcccta acggttggga agatttgact 600
gtcggtggtc cccttgattt tccgacacca tccgagccga tgacacctct gagtgagcct 660
gtgcttatgc ccgcgtcgca acatatttct aggccagtga cacctttgag tgggccggcc 720
ccagttcctg caccgcgtag aactgtgtcc cgaccgatga cgcccttgag tgagccaatt 780
tttgtgtctg caccgcgaca caaatttcag caggtggaag aagcgaatcc ggcggcaaca 840
acgctgacgt atcaggacga gcctctagat ttgtctgcct cctctc 886
<210> 4
<211> 796
<212> DNA
<213>PRRSV genes
<400> 4
gacacctcct ttgattggga cgttgtgttt cctggggttg aggcggcaaa tcagaccgcc 60
gaacaacctc acgccaactc atgctgtacc ctagtccctc ccgtgactca agagcctctg 120
ggcaaggact cggtccctct gaccgccttc tcactgtcca atcgctatta ccctgcacaa 180
ggcgacgagg ttcatcaccg tgagaggcta aattctgtac tctctaagtt ggaagaggtt 240
gtcctggaag aatatgggtt catgcccact ggatttggcc cgcgacctgt gctgccgagc 300
gggctcgacg agcttaaaga ccggatggag gaggatctgc taaaactagc caacacccag 360
gcgacttcag aaatgatggc cagggcggcc gagcaggtcg atttaaaaac ttgggttaaa 420
agctacccgc ggtggacacc accacccctt ccaccaagag ttcaacctcg aagaacaaag 480
tctgcaaaaa gcttgccaga gggcaagcct gtcccagctc cgcgtaggaa ggtcagatcc 540
gactgcggca gtcctgcctg gatgggcaac aatgtgccta acagttcgga agaaactgtc 600
ggcgaacccc ccaattttcc gacaccagcc gagctgatga cacctgttgg tgagcccgta 660
cttgtgcctg cgttgcaaca tgtccccaag ctaatgacac ctttgagtgg gtcggcacca 720
gttcctgcac cgcgtagaac tgttacaaca acgctgacgc accaggacga gcctctggat 780
ttgtctgcct cctctc 796
<210> 5
<211> 493
<212> DNA
<213>PRRSV genes
<400> 5
gacacctcct ttgattggga tttcgtgctg cctggagttg gagtggctgc tcaagcagcg 60
gaattgcccc ccatcagcca gtgtcacgct cctgtcaccg ttgtagccca aaggtcttcg 120
ccggaattcc agtctcggaa agcggagtct gtcaggagcc ttccagagaa caggcctctt 180
cctgccccac gcaggaagat caggtccagg tgtggtagtt tggtttcatt gggcggcaac 240
ttccctgaca gctgggtaga cttggtcggt ggttccttcc attccccagt cctgcctgag 300
tcagtggcac gttcgaatga gcctgtgcct gtcccggcgc cacgcaggat tgtgccccgg 360
ctgaagccgt caccaataac accaactcct gtgcccgcgc cacgatctgg gcttcagcag 420
gtgggaggaa tgaacttagc ggtagggact ctggcgtgcc aggacgagct cctcgatttg 480
tctgcctcct ctc 493
<210> 6
<211> 20
<212> DNA
<213>Artificial sequence
<400> 6
ggttcggaag aaactgtcgg 20
<210> 7
<211> 21
<212> DNA
<213>Artificial sequence
<400> 7
agcaggtgga agaagcgaat c 21
<210> 8
<211> 21
<212> DNA
<213>Artificial sequence
<400> 8
gagctgagta ttttgggcgt g 21
Claims (6)
1. it is a kind of to detect pig blue-ear disease poison classical strainses, the primer of highly pathogenic mutant strain and NADC-30 strains, for RT-
PCR reacts, it is characterised in that the primer includes the nucleosides of sense primer and anti-sense primer, the sense primer and anti-sense primer
Acid sequence is respectively such as SEQ ID NO:1 and SEQ ID NO:Shown in 2.
2. it is a kind of to detect pig blue-ear disease poison classical strainses, the RT-PCR method of highly pathogenic mutant strain and NADC-30 strains, its
It is characterised by, comprises the following steps：
As template, reverse transcription obtains cDNA to RNA with detected sample, with the primer described in claim 1, to sample to be tested
RT-PCR amplifications are carried out, amplified production is detected with 1.5% agarose gel electrophoresis；Amplified production size is 887bp, then in sample
Containing or candidate contain pig blue-ear disease poison classical strainses；Amplified production size is 797bp, then contain in sample or candidate contains height
Pathogenicity variation strain；Amplified production size is 493bp, then contain in sample or candidate contains NADC-30 strains.
3. it is according to claim 2 to detect pig blue-ear disease poison classical strainses, highly pathogenic mutant strain and NADC-30 strains
RT-PCR method, it is characterised in that inverted with the reverse transcription reagent box purchased from precious bioengineering (Dalian) Co., Ltd
Record, be the step of the reverse transcription：By the μ L of RNA templates 4 and No. 1 the μ L of 5 × Prime Script Buffer 4, No. 2 Prime
The μ L of ScriptRT Enzyme Mix 1, No. 4 μ L of Random 6mers 2 and No. 5 RNase Free dH2The μ L of O 9 are mixed, and are put into
Reverse transcription is carried out in PCR instrument.
4. it is according to claim 2 to detect pig blue-ear disease poison classical strainses, highly pathogenic mutant strain and NADC-30 strains
RT-PCR method, it is characterised in that the reaction condition of the reverse transcription is：37 DEG C of 15min, 85 DEG C of 15s, 16 DEG C of 10min.
5. it is according to claim 2 to detect pig blue-ear disease poison classical strainses, highly pathogenic mutant strain and NADC-30 strains
RT-PCR method, it is characterised in that the reaction system of RT-PCR amplification is as follows：Template cDNA 3 μ L, Quick Taq
The μ L of HS DyeMix 10, sense primer and anti-sense primer each 0.6 μ L, it is remaining by ddH2O is supplied, and total amount is 20 μ L.
6. it is according to claim 2 to detect pig blue-ear disease poison classical strainses, highly pathogenic mutant strain and NADC-30 strains
RT-PCR method, it is characterised in that the reaction system condition of RT-PCR amplification is as follows：94 DEG C of predegeneration 2min；94℃
Denaturation 30s, 55 DEG C of annealing 30s, 68 DEG C of extension 1min, totally 35 circulations；72 DEG C of extension 8min.
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| CN107502679A (en) * | 2017-09-21 | 2017-12-22 | 南京农业大学 | Differentiate LAMP primer and discrimination method and the application of PRRSV strain gene hypotypes |
| CN109762933A (en) * | 2019-01-25 | 2019-05-17 | 长江大学 | A kind of universal pig blue-ear disease poison triple nide RT-PCR detection primer and method |
| CN110885900A (en) * | 2018-09-10 | 2020-03-17 | 中国动物疫病预防控制中心（农业部屠宰技术中心） | Freeze-drying microchip, kit and method for identifying classical strains of Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) and strain NADC30-Like |
| CN110885902A (en) * | 2018-09-10 | 2020-03-17 | 北京亿森宝生物科技有限公司 | Freeze-drying microchip, kit and method for detecting porcine reproductive and respiratory syndrome virus and identifying highly pathogenic classical variant strain of porcine reproductive and respiratory syndrome virus |
| CN112094952A (en) * | 2020-10-28 | 2020-12-18 | 云南农业大学 | Complete set of primer pair for porcine reproductive and respiratory syndrome virus whole genome determination and application thereof |
| CN114292961A (en) * | 2022-01-29 | 2022-04-08 | 龙岩学院 | Real-time fluorescent RT-PCR detection primer and probe set for identifying NADC-30-like strains |
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| CN107502679A (en) * | 2017-09-21 | 2017-12-22 | 南京农业大学 | Differentiate LAMP primer and discrimination method and the application of PRRSV strain gene hypotypes |
| CN110885900A (en) * | 2018-09-10 | 2020-03-17 | 中国动物疫病预防控制中心（农业部屠宰技术中心） | Freeze-drying microchip, kit and method for identifying classical strains of Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) and strain NADC30-Like |
| CN110885902A (en) * | 2018-09-10 | 2020-03-17 | 北京亿森宝生物科技有限公司 | Freeze-drying microchip, kit and method for detecting porcine reproductive and respiratory syndrome virus and identifying highly pathogenic classical variant strain of porcine reproductive and respiratory syndrome virus |
| CN110885902B (en) * | 2018-09-10 | 2022-10-21 | 北京亿森宝生物科技有限公司 | Freeze-drying microchip, kit and method for detecting porcine reproductive and respiratory syndrome virus and identifying highly pathogenic classical variant strain of porcine reproductive and respiratory syndrome virus |
| CN110885900B (en) * | 2018-09-10 | 2023-08-22 | 中国动物疫病预防控制中心（农业部屠宰技术中心） | Freeze-dried microchip, kit and method for identifying classical strain of porcine reproductive and respiratory syndrome virus and NADC30-Like strain |
| CN109762933A (en) * | 2019-01-25 | 2019-05-17 | 长江大学 | A kind of universal pig blue-ear disease poison triple nide RT-PCR detection primer and method |
| CN112094952A (en) * | 2020-10-28 | 2020-12-18 | 云南农业大学 | Complete set of primer pair for porcine reproductive and respiratory syndrome virus whole genome determination and application thereof |
| CN114292961A (en) * | 2022-01-29 | 2022-04-08 | 龙岩学院 | Real-time fluorescent RT-PCR detection primer and probe set for identifying NADC-30-like strains |
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